中药虎杖雌激素活性组分的分离及活性测试

药材虎杖用70%乙醇冷浸法进行提取。对虎杖中蒽醌类化合物集中分布的乙酸乙酯分配组分首先进行硅胶柱分离,得到6个组分,利用转基因酵母法测定其雌激素活性,结果表明前三个组分具有较高的雌激素活性。进一步对其中的第三个活性组分利用制备高效液相色谱进行了精制,对流出物按2分钟间隔收集,共得到20个馏分。转基因酵母法测定其雌激素活性的结果显示,在这20个馏分当中,有5个馏分具有较强的雌激素活性。分别将其雌激素活性与化学组成进行关联,发现其中的sub-fraction-13中可能存在非大黄素的雌激素活性成分。     

虎杖MYB转录因子PcMYB1的表达特性和功能研究

目的对虎杖中1个新的R2R3-MYB转录因子基因PcMYB1进行转录活性鉴定和表达特性分析,并在转基因拟南芥中进行功能研究。方法利用酵母单杂交实验分析PcMYB1的转录活性;荧光定量PCR(RT-PCR)技术检测虎杖中PcMYB1的表达模式;利用Wiesner染色和溴乙酰法检测PcMYB1转基因拟南芥中的木质素含量;RT-PCR技术分析PcMYB1转基因拟南芥木质素合成相关基因的表达。结果酵母单杂实验结果表明,PcMYB1具有转录抑制活性;RT-PCR结果显示PcMYB1在虎杖根、茎、叶中均有表达,在叶中表达量最高,并且紫外照射处理可诱导叶片中PcMYB1的表达;与野生型拟南芥相比,转基因拟南芥的株高降低了24.07%,木质部的细胞染色程度浅,转基因拟南芥木质素含量降低了14.81%,参与木质素合成的AtC4H、AtC3H、AtF5H、AtCOMT、AtCAD基因表达下调。结论 PcMYB1具有转录抑制活性,对植物木质素合成具有负调控作用。     

基于LC-MS的蛇倒退抗肿瘤活性部位筛选及鉴定研究

目的:分离得到蛇倒退中抗肿瘤活性部位并鉴定。方法:通过硅胶柱色谱分离蛇倒退得到不同馏分，以SGC-7901的抑制率和IC₅₀为指标筛选得到抗肿瘤作用最强部位，并在倒置显微镜下观察细胞形态及应用LC-MS对其进行鉴定。结果:经分离得到4个主要馏分，其中馏分1的抗肿瘤活性最强，LC-MS鉴定得到一系列化合物。结论:蛇倒退中分离得到的PEC A部位具有良好的抗肿瘤活性，其主要成分为黄酮类化合物。     

狼毒大戟抗肿瘤活性成分

目的:筛选狼毒大戟抗肿瘤活性馏分,分离活性单体化合物并对构效关系进行探讨。利用超高效液相色谱-质谱联用技术(UPLC-Q-TOF-MS)对活性馏分的化学成分进行鉴定。方法:采用噻唑蓝(MTT)法对狼毒大戟乙醇提取物的石油醚、乙酸乙酯、正丁醇和水萃取部位的抗肿瘤活性进行测试。综合运用多种现代色谱分离手段对活性萃取层石油醚层进行分离。利用核磁(NMR)和质谱(MS)技术鉴定化合物的化学结构。质谱使用ESI离子源,正离子模式下采集数据,结合狼毒大戟相关文献以及对照品信息进行数据分析,解析活性馏分的化合物结构。结果:从活性馏分层石油醚层分离得到6个单体化合物,分别为岩大戟内酯A,岩大戟内酯B,17-羟-岩大戟内酯A,17-羟-岩大戟内酯B,euphopilolide,atis-16-en-13(s)-hydroxy-3,14-dione。对这些化合物的抗肿瘤活性的构效关系进行了探讨。利用UPLC-Q-TOF-MS对狼毒大戟乙醇提取物的石油醚萃取部位化学成分进行鉴定,共鉴定化合物23个,其中二萜类成分19个,酚类成分2个,脂肪酸类成分1个,三萜类成分1个。峰18和峰21初步鉴定为新化合物。结论:狼毒大戟的活性馏分层为石油醚层,其主要成分为二萜类,具有较好的抗肿瘤活性。UPLC-Q-TOF-MS技术能够快速准确地鉴定狼毒大戟活性馏分的化学成分结构,为其质量评价和活性物质基础研究提供了参考。     

天花粉降血糖活性成分的分离和活性观察

目的对天花粉降血糖的活性成分进行分离和活性观察。方法用醇超声、醇回流、水超声提取方法制备3种天花粉提取物,以四氧嘧啶糖尿病小鼠模型对其降血糖的活性进行考察,对其中活性较强者进一步通过大孔吸附树脂和硅胶柱色谱进行活性成分分离,并通过细胞模型计算和比较其对细胞葡萄糖的消耗量。结果醇超声提取方法具有良好的降血糖功效及改善糖尿病"三多一少"症状,其中80%洗脱组分对细胞葡萄糖消耗量相对较大,与模型组及其它洗脱组分比较具有显著差异(P<0.001);进一步分离得到化合物Ⅰ、Ⅱ、Ⅲ,其中化合物Ⅰ对细胞葡萄糖消耗量与模型组比较具有显著差异(P<0.05)。结论醇超声提取物80%洗脱组分为天花粉降血糖的活性成分,其中化合物Ⅰ可能为天花粉降血糖的一个活性化合物,具体结构有待进一步深入探讨。     

虎杖查耳酮合酶基因RNAi载体的构建及其遗传转化

目的构建虎杖查耳酮合酶(Pc CHS1)基因的RNA干涉(RNAi)表达载体,获得Pc CHS1表达下调的转基因虎杖植株。方法根据Gen Bank中已知的Pc CHS1基因序列(EF090604),设计相应引物,克隆Pc CHS1基因核心保守序列。以Pc CHS1基因为靶基因,将长度574 bp保守序列片段通过正、反2个方向插入表达载体p YLRNAi中,构建RNAi表达载体p YLRNAi-Pc CHS1。通过根癌农杆菌介导法将其导入虎杖茎尖组织。对获得的转基因植株,利用Northern blotting检测Pc CHS1基因表达水平,并应用HPLC法测定虎杖中白藜芦醇苷的量。结果成功构建Pc CHS1基因RNA干涉表达载体,获得了5株转Pc CHS1基因干涉载体的阳性植株。转基因虎杖Pc CHS1基因表达水平显著下调,并且转基因植株中白藜芦醇苷量均得到显著提高,其中最高量是对照植株的3.8倍(P0.05)。结论成功获得了干涉Pc CHS1基因表达下调的转化植株,抑制Pc CHS1基因的表达显著增加了转基因虎杖中白藜芦醇苷的量,为有效利用该基因提高虎杖白藜芦醇苷量奠定基础。     

虎杖叶的化学成分、药理活性、临床应用及质量控制研究进展

虎杖叶药用历史悠久,资源分布广,蕴藏量大。虎杖叶的主要化学成分包括黄酮、蒽醌和酚酸类,此外还有二苯乙烯及萘酚类等成分,其中黄酮是其主要活性成分。现代药理研究表明,虎杖叶中多酚类具有抗氧化活性,黄酮类还具有降血压、血糖及血脂等活性。虎杖叶在临床上主要用于肝阳上亢导致的眩晕,常见的头晕、头昏、头痛等症。对虎杖叶的化学成分、药理作用和临床应用及质量控制进行综述,为其开发利用提供参考。     

虎杖靶向富集活性组分抗HIV-1活性研究

目的探讨虎杖Polygonum cuspidatum富集活性组分体外对人类免疫缺陷病毒1型(HIV-1)的抑制作用及作用靶点。方法运用表面等离子共振HIV-1多靶点筛选系统对虎杖不同工艺提取物进行筛选,整合酶氨基耦联柱靶向富集提取物中高活性成分,在TZM-bl细胞和PBMC细胞中对富集产物进行抗HIV-1病毒活性评价;采用荧光共振能量转移分析法测定药物对HIV-1整合酶3’加工的抑制作用;运用高通量ELISA法测定虎杖60%乙醇提取物的整合酶靶向富集产物(HZ60-IN)对整合酶链转移的影响;试剂盒检测HZ60-IN对逆转录酶和蛋白酶的影响。结果 HZ60-IN对整合酶有高亲和性。细胞水平病毒感染实验表明,在TZM-bl细胞中,HZ60-IN对HIV-1 NL4.3和1084i的半数抑制浓度(IC_(50))分别为(31.94±8.96)、(38.07±11.25)μg/mL;在2株PBMC细胞中,HZ60-IN对HIV-1 NL4.3病毒均显示显著的抑制活性。HZ60-IN对整合酶的3’加工和链转移均有抑制作用,其中对3’加工的IC_(50)为(6.54±1.69)μg/mL,对链转移的IC_(50)为(2.56±0.97)μg/mL,其不作用于HIV感染的进入阶段,对逆转录酶抑制活性较弱,对蛋白酶活性没有影响。结论 HZ60-IN有抗HIV-1病毒活性,主要通过影响HIV-1的整合酶活性发挥作用。     

中药虎杖中抗癌活性物质研究

目的:为虎杖药用价值的开发利用提供科学据依。方法:在生物活性测定指导下,从虎杖中分离得到一种较高抗癌活性的物质,并且用液质联用、红外光谱、紫外全扫描等方法鉴定其结构。MTT法测定它对L-02、HepG2、SHZ-888、MCF-7、MCF-7/ADM等细胞的抑制作用并用HE染色法观察其作用MCF-7/ADM细胞48 h、72 h后的肿瘤细胞形态学变化。结果:分离得到的物质为顺、反式-白藜芦醇,它能特异性抑制多种肿瘤细胞的生长,而对正常肝细胞毒性很小。同时,首次发现其对耐阿霉素的MCF-7乳腺癌细胞有直接的细胞毒性。结论:白藜芦醇是虎杖中一种活性强、低毒的抗癌物质。     

虎杖及大黄中的雌激素样活性成分:蒽醌类的结构与活性

在对数种生药的甲醇提取物筛选有效成分时发现,虎杖(Polygonum cuspidatum)及药用大黄(Rheum officinalis)提取物有强活性,并且发现了大黄素等蒽醌成分。为了明确蒽醌类的雌激素样活性的构效关系,对各种衍生物的活性进行了探讨。 实验与结果:观察虎杖、好望角芦荟、     

黑大豆乙醇提取物的雌激素样作用及其机制研究

目的:评价黑大豆乙醇提取物(black soybean ethanol extract,BSE)的雌激素样作用及其作用机制。方法:采用富含雌激素受体的雌激素依赖性人乳腺癌细胞株MCF-7细胞,分别以E-SCREEN法观察BSE对其增殖的影响、以RT-PCR法观察BSE对其雌激素效应基因mRNA表达的影响,并以雌激素受体拮抗剂ICI182,780为工具药来评价BSE发挥雌激素样作用的机制。结果:与溶剂对照组相比较,BSE(10～200μg.mL^-1)能显著促进MCF-7细胞的增殖,100μg.mL^-1时作用达到最大;BSE能明显诱导MCF-7细胞pS₂,孕激素受体(PR)基因mRNA的表达,并与药物浓度有一定相关性;以上作用均能被雌激素受体拮抗剂所完全拮抗。结论:BSE能促进MCF-7细胞的增殖,诱导雌激素效应基因mRNA的表达,即BSE能通过雌激素受体发挥雌激素样作用。     

刺梨多糖的分离纯化及其神经营养活性

 目的对刺梨多糖进行分离纯化及神经营养活性研究。方法采用DEAE-纤维素柱色谱及Sepharose CL-6B凝胶柱色谱对刺梨粗多糖进行分离纯化,得到RRTP-1～RRTP-8共8个多糖组分。凝胶柱色谱和聚丙烯酰胺凝胶电泳鉴定纯度。采用PC12细胞对各组分的神经营养活性进行筛选,并对纯化的单一多糖进行活性定量测定。结果RRTP-1和RRTP-3为相对分子质量分布均一多糖。PC12细胞实验结果表明,多个多糖组分能刺激PC12细胞产生神经纤维样突起。结论多数刺梨多糖组分具有神经营养活性。     

Analysis of estrogenic compounds in Polygonum cuspidatum by bioassay and high performance liquid chromatography

The estrogenic activity of traditional Chinese herb-Polygonum cuspidatum Sieb. et Zucc. was investigated by a recombinant yeast screening (YES) assay. Anthraquinones are the main components in the plant, of which emodin is the most abundant one. The ethyl acetate fraction of the ethanol extract of Polygonum cuspidatum was separated on a silica gel TLC plate and seven sub-fractions were collected. The results of bioassay demonstrated that Hzs1 and Hzs6 showed higher estrogenic activities than that of others and the potency of these two compounds were approximately 10(-4) g/L and 10(-3) g/L, respectively. HPLC analysis was performed to determine the activities and the active components. Combining the results of HPLC analysis and estrogenic activity test by YES led to the conclusion that an unknown bioactive compound might exist in the extraction of Polygonum cuspidatum.     

赤豆的雌激素样作用及其对人类乳腺癌MCF-7细胞孕激素受体水平的影响

目的：评价赤豆的雌激素样作用及其对人类乳腺癌MCF-7细胞孕激素受体水平的影响。方法：采用富含雌激素受体的雌激素依赖性人乳腺癌细胞株MCF-7细胞，分别以E-SCREEN法观察赤豆提取物对其增殖的影响，以RT-PCR法、Western blot法观察赤豆对孕激素受体mRNA和蛋白表达的影响，并以雌激素受体拮抗剂ICI182,780为工具药来评价赤豆提取物发挥雌激素样作用的机制。结果：与溶剂对照组比较，赤豆（10～200 μg·mL^-1）能显著促进MCF-7细胞的增殖，100 μg·mL^-1时作用达到最大；赤豆提取物能明显诱导MCF-7细胞孕激素受体(PR)基因mRNA和蛋白的表达，以上作用均能被雌激素受体拮抗剂所完全拮抗。结论：赤豆提取物具有雌激素活性，此作用是通过雌激素受体(ER)介导的。     

Estrogenic activity of Nigella damascena extracts,evaluated using a recombinant yeast screen

We used the yeast estrogen screen (YES) containing a human estrogen receptor to evaluate the estrogenic activity of extracts obtained from Nigella damascena seeds. Alcohol extracts obtained by direct extraction of seeds showed a low estrogenic activity, while the alcohol extract obtained after extraction with solvents of increasing polarity showed a strong estrogenic activity. This suggests the presence in Nigella of polar components whose activity can be clearly demonstrated after previous elimination of interacting apolar components that may mask the activity of more polar components. The response of both alcohol fractions follow a bell-shaped curve indicating a concentration-dependent relationship.     

Post-coital contraceptive effects of Ferula jaeschkeana Vatke extracts in rats and hamsters and their hormonal properties

Various extracts of Ferula jaeschkeana Vatke have been studied for their anti-implantation activity in rats and hamsters along with an assessment of hormonal properties like estrogenic, antiestrogenic, progestagenic and antiprogestagenic actions. Of the various extracts produced, hexane, benzene and chloroform fractions prevented implantation in all the rats when administered at 25 mg/kg dose for 7 days after coitus. These fractions were ineffective in golden hamsters. In the hormonal studies, the hexane extract increased significantly the uterine wet weight in bilaterally ovariectomized rats and also stimulated the histological features of the uterus. Its estrogenic activity has also been confirmed by its capacity to induce implantation in rats undergoing experimentally delayed implantation. Its conjoint administration with diethylstilbesterol showed a synergistic effect. None of the doses of the hexane extract could interfere with the action of standard progesterone when tested in mature and immature rats. The anti-implantation activity of the extracts was considered to be due to its frank and potent estrogenic action.     

Susceptibility of Helicobacter pylori to essential oil of Dittrichia viscosa subsp. revoluta

The essential oil of Dittrichia viscosa subsp. revoluta and its fractions were assessed for anti-Helicobacter activity. The essential oil was isolated by hydrodistillation, submitted to flash column chromatography and analysed by gas chromatography, gas-chromatography coupled to mass spectrometry and (13)C-nuclear magnetic resonance. The anti-Helicobacter activity was determined by incorporation of the crude essential oil and oxygenated fractions of the oil into the culture medium. At a concentration of 0.025 microL/mL no recovery was registered when one of the oxygenated fractions of the oil, mainly constituted by 3-methoxy cuminyl isobutyrate (about 40%), was used. This fraction revealed a higher activity against the six H. pylori strains tested when compared with the other oxygenated fractions. The crude essential oil at a concentration of 0.33 microL/mL reduced the initial population of H. pylori CCUG 15818 of 8.52 +/- 0.30 log(10) cfu/mL to 7.67 +/- 0.22 log(10) cfu/mL. The susceptibility of several Helicobacter pylori strains to the oxygenated fraction of Dittrichia viscosa subsp. revoluta essential oil suggests the possible use of these natural products in combating this widespread infection.     

重组雌激素受体基因酵母法对南山茶子抗骨质疏松活性部位的研究

目的:明确南山茶子抗骨质疏松的有效部位。方法:用系统溶剂分离法将南山茶子醇提物分成极性不同的组分,利用重组人雌激素受体基因酵母生物检测法筛选山茶抗骨质疏松的有效部位。结果:5个组分都表现出类雌激素和抗雌激素的双重效应,活性强弱依次为乙酸乙酯层>醇提物>正丁醇层>石油醚层>水层。结论:乙酸乙酯部位为山茶抗骨质疏松的有效部位。     

A promising peptide antibiotic from Terfezia claveryi aqueous extract against Staphylococcus aureus in vitro

The antimicrobial activity of aqueous and methanolic extracts, as well as partially purified proteins extracted from Terfezia claveryi aqueous extract were investigated against Staphylococcus aureus in vitro. A 5% aqueous extract inhibited the growth of S. aureus by 66.4%, while a methanolic extract was ineffective. Partial protein purification of the aqueous extract using ammonium sulphate precipitation revealed that antimicrobial activity was within the third fraction. This fraction was then subjected to gel filtration using Sephadex G-100. Two peaks were obtained. Peak one possessed higher antimicrobial activity. This peak was then subjected to ion exchange chromatography using DEAE Sephadex. Only peak 4 from the six peaks obtained showed a slight antimicrobial activity. Antimicrobial activities of the aqueous extract and the fractions that showed antimicrobial activity were compared with reference antibiotics.